KR101569813B1 - Composition for preventing or treating brain disease or neural disease comprising oriental herbal extracts - Google Patents

Abstract

The present invention relates to a composition for preventing or treating a brain disease or a neurological disease, which comprises an effective component of a combined extract of greenhouse ginseng, guinea fowl, groundnut, and Seokchangpo. The complex extract according to the present invention protects hippocampal cells, increases proliferation of neural stem cells, inhibits hyperphosphorylation of tau protein, increases cerebral blood flow in animal models, alleviates cerebral infarction, Improving ability, and can be useful for prevention or treatment of brain diseases or neurological diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating a brain disease or a neurological disease comprising an extract of a herbal medicine as an active ingredient,

The present invention relates to a composition for preventing or treating a brain disease or a neurological disease, which comprises an effective component of a combined extract of greenhouse ginseng, guinea fowl, groundnut, and Seokchangpo.

In modern life, memory is becoming increasingly important in rapidly changing lives, and it has become a major interest from young people with a lot of social skills to old age. The deterioration of these functions is a social problem, and in the society where the proportion of the elderly population is increasing, the deterioration of memory in the possibility of social life is very important factor in patients who are relaxed to degenerative diseases such as dementia. Dementia is the fourth cause of cancer deaths, heart disease and stroke following death, which greatly reduces the quality of life, both personally and socially, and makes people and people around them unhappy. Dementia is divided into Alzheimer's dementia and vascular dementia. Alzheimer's dementia is a type of dementia observed in western countries. It is a disease in which cholinergic neurons are destroyed by accumulation of beta-amyloid in specific parts of the brain. Vascular dementia is a dementia caused by hypertension, stroke, hyperlipidemia, etc., resulting in ischemic conditions due to abnormal blood vessels to the brain, resulting in death in the hippocampus and peripheral nervous system. These two types of dementia have different mechanisms, but they have the same mechanism in that they reduce the action of acetylcholine, which is known as a memory-transmitting neuron, resulting in impaired memory.

Efforts have been made to develop various therapeutic strategies to improve and improve the cognitive function and learning function deteriorated due to recent dementia and to develop effective drugs. There are acetylcholine precursors, receptor agonists, acetylcholine esterase inhibitors, and so on. However, there have not been developed therapeutic agents capable of treating the underlying cause of Alzheimer's disease so far. Common therapeutic agents include Aricept, an acetylcholinesterase inhibitor of Pfizer, Exelon of Novartis, Reminyl, and recently Ebixa (Memantine) from Lundbeck, an antagonist of NMDA receptors licensed from the US FDA. However, acetylcholinesterase inhibitors improve the declining cognitive ability and do not cure the underlying cause of Alzheimer's disease. In addition, it is difficult to say that it is a fundamental treatment agent because it shows a temporary symptomatic relief effect only in some patients and its effect does not last long. In addition, due to the nature of the disease, a long-term use is required. In the case of the above-mentioned medicines, accompanying toxic effects, vomiting, loss of appetite, Therefore, it is urgent to develop a therapeutic agent that can prevent the progress of the disease. To this end, many multinational pharmaceutical companies have invested heavily in R & D in this field. In particular, beta or gamma secretase inhibitors, which reduce the production of beta amyloid consisting of about 40 amino acids, which is presumed to be a fundamental cause of Alzheimer's disease Is the main type of development. In Korea, basic research on Alzheimer's disease has been done to some extent, but the development of dementia treatment itself is rarely done.

Accordingly, the inventors of the present invention searched for herbal medicines in order to develop a therapeutic agent capable of effectively treating brain diseases and neurological diseases. As a result, it has been found that a combination extract of Hashuo, Hwanghwang, Sajang, and Seokchangpo protect hippocampal cells and increase proliferation of neural stem cells And inhibited hyperphosphorylation of tau protein. It was confirmed that the animal model increases cerebral blood flow, alleviates cerebral infarction, improves memory, cognitive ability, and learning ability, and has accomplished the present invention.

An aspect of the present invention is to provide a pharmaceutical composition and a food composition for preventing or treating a brain disease or a neurological disease, which comprises, as an active ingredient, a combined extract of red sea bream,

An aspect of the present invention provides a composition for the prevention or treatment of brain diseases or neurological diseases, which comprises, as an active ingredient, a combined extract of red algae, guinea pigs, groundnut, and Seokchangpo.

The composition includes a pharmaceutical composition and a food composition.

Hereinafter, the present invention will be described in detail.

As an active ingredient of the present invention, " red algae " is a dried root of Polygonum multiflorum with Asclepiadaceae . Also, as an active ingredient of the invention "Rehmannia" is Rehmannia of the Scrophulariaceae (Rehmannia glutinosa ). As an active ingredient of the present invention, "sheet" is the scientific name a perennial dicotyledonous geraniales polygalaceae is Polygala tenuifolia . Also, as an active ingredient of the invention "seokchangpo" nomenclature is a perennial plant of the monocotyledonous plants are Araceae neck Araceae Acorus gramineus .

The complex extract according to the present invention can be extracted by the following method. First, measure the red mulberry, red mulberry, red mulberry, and silkworm, and wash them with high pressure spraying. Mix the washed raw materials and extract by adding an appropriate amount of solvent. The extraction can be impregnated or warmed at room temperature. Preferably by hot water extraction. The solvent may be water, an alcohol having 1 to 5 carbon atoms, acetone, ethyl acetate, butyl acetate, chloroform, hexane, diethyl ether, 1,3-butylene glycol and mixtures thereof. Preferably, have. The step of concentrating or filtering the extract may be further treated.

In the composition of the present invention, the mixing ratio of the above-mentioned water, sulfur, persimmon, ground and sea bream may be 1 to 20: 0.5 to 10: 0.1 to 5: 0.1 to 5 by weight. More preferably 1 to 10: 1 to 5: 0.5 to 3: 0.5 to 3, and most preferably 5.1: 1.9: 1.5: 1.5.

The complex extract according to the present invention protects hippocampal cells, increases proliferation of neural stem cells, inhibits hyperphosphorylation of tau protein, increases cerebral blood flow in animal models, alleviates cerebral infarction, Improving ability, and can be useful for prevention or treatment of brain diseases or neurological diseases.

Therefore, the combined extract of greenhouse ginseng, guinea pig, raw paper and Seokchangpo of the present invention can be used as a pharmaceutical composition for preventing or treating brain diseases or neurological diseases.

The brain disease may be stroke, stroke, stroke, cerebral infarction, traumatic brain injury, shock brain damage, hypoxic brain injury, ischemic brain disease, or degenerative brain disease.

The neurological diseases may also be selected from the group consisting of dementia, Alzheimer's disease, Parkinson's disease, stroke, Huntington's disease, Kruszfeld-Jjark's disease, Pick's disease, amyotrophic lateral sclerosis, Parkins-ALS-dementia complex, Wilson's disease, Cognitive impairment, sclerosis, progressivesupranuclear palsy, mild cognitive impairment or epilepsy.

In particular, when the composition is used as a food composition, it can be used for enhancing brain or cognitive function, which may be memory, thinking, comprehension, calculation, learning, judgment or concentration.

In addition, the brain disease or neurological disease may be a memory disorder, a cognitive disorder, or a learning disorder, and such memory, cognition, and learning disorder may be caused by various causes and environments, and the cause thereof is not limited. For example, there is provided a method of treating a condition selected from the group consisting of aging, Alzheimer's disease, schizophrenia, Parkinson's disease, Huntington's disease, Peak disease, Creutzfeldt-Jakob disease, depression, aging, head trauma, stroke, CNS hypoxia, , Cerebral vasculopathy, cerebral vascular disease, cardiovascular disease, amnesia, cerebral palsy, cerebral apoplexy, headache, brain aging, dementia, anterior temporal lobe degeneration, , Radiation exposure, metabolic disease, hypothyroidism, mild cognitive impairment, memory impairment due to cognitive deficit and attention deficit, cognitive impairment or learning impairment.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the composition include those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. Upon oral administration, the oral composition is preferably formulated so as to coat the active agent or protect it from degradation at the top. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition may be variously prescribed depending on factors such as the formulation method, the administration method, the patient's age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness have. The preferred dose of the composition is in the range of 0.001-100 mg / kg on an adult basis. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat a memory disorder, cognitive disorder, or learning disorder.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art, or may be prepared by inserting it into a multi-dose container . The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent.

In addition, one aspect of the present invention provides a composition for foods for preventing or ameliorating brain diseases or neurological diseases and a food composition for enhancing brain or cognitive function, which comprises the above-described complex extract as an active ingredient. The brain or cognitive function may be memory, thinking, comprehension, computing ability, learning ability, judgment or concentration. In this case, the amount of the complex extract in the food or beverage may be 0.01 to 15% by weight of the total food, and the health drink composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, But it can be easily determined by a person skilled in the art to suit the product.

The food composition may further comprise a pharmaceutically acceptable food-aid additive in addition to the complex extract, and may be prepared in the form of tablets, capsules, pills, solutions, and the like.

The food composition of the present invention is not particularly limited as long as it contains the above-mentioned complex extract as an essential ingredient, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

The food composition of the present invention may contain other various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The ratio of such additives is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

The complex extract according to the present invention protects hippocampal cells, increases proliferation of neural stem cells, inhibits hyperphosphorylation of tau protein, increases cerebral blood flow in animal models, alleviates cerebral infarction, Improving ability, and can be useful for prevention or treatment of brain diseases or neurological diseases.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a process for producing a complex extract of the present invention. FIG.
Fig. 2 is a result of high-performance liquid chromatography analysis of the main functional components of the red mud (A) and the combined extract (B). Separation was carried out on a Luna C18 (2) column (Phenomenex Inc., Torrance, Calif.).
FIG. 3 shows the protective effect of the complex extract on hippocampal cell HT22.
Fig. 4 is a graph showing the effect of the complex extract on the proliferation of neural stem cells.
FIG. 5 is a graph showing the effect of the combined extract on cerebral cortex inhibition of tau protein hypoxia.
FIG. 6 is a graph showing the effect of the combined extract on cerebral blood flow in the middle cerebral artery region. FIG.
FIG. 7 is a graph showing the effect of the combined extract on cerebral infarction and edema in the model of the mineral oil-induced thrombosis.
FIG. 8 is a graph showing the effect of dietary supplementation on the body weight of experimental animals.
FIG. 9 shows the effect of the combined extract on the total travel distance of the underwater labyrinth analysis. FIG.
10 shows the effect of the combined extract on swim speed.
FIG. 11 shows the effect of the complex extract on the time of finding the hidden platform. FIG.
12 shows the effect of the combined extract on the delay time of the passive avoidance assay.
13 shows the effect of the combined extract on the performance of the dementia model.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Preparation of complex extract

The combined extracts of Korean red ginseng, Chinese ginseng, Chinese ginseng, and Chinese ginseng were compounded as shown in Table 1 below.

School name Scientific name Mixing ratio Enemy Polygonum multiflorum 5.1% Rehab Rehmannia glutinosa 1.9% Origin Polygala tenuifolia 1.5% Seokchangpo Acorus gramineus 1.5% Purified water 90.0% total 100%

The raw materials passed in the inspection of the stocking were weighed according to the above mixing ratio and then subjected to high pressure spray washing. After the washed raw materials were mixed, purified water of 9 times the weight of the raw material was added, and the mixture was subjected to hot water extraction at 120 ± 5 ° C. for 4 hours or more and concentrated at a low temperature of 70 ± 5 ° C. at a brix of 20% or more. The concentrated concentrate was dried in a spray drier at an input temperature of 180 ± 5 ° C and a discharge temperature of 105 ± 5 ° C, followed by pulverizing the dried material to prepare a powder. 1 shows a process for producing the complex extract of the present invention.

Example  2: High Performance Liquid Chromatography of Compound Extracts ( HPLC ) analysis

In the combined extracts, 2,3,5,4'-tetrahydroxystilbene-2-O (2,3,5,4'-tetrahydroxystilbene-2-O The content of stilbeneglycoside, which is β-D-glucoside, was compared. The results are shown in FIG. 2 and Table 2 below.

ingredient Content (mg / 100 mg extract) Enemy Complex extract 2,3,5,4 ' -tetrahydoxystilbene-2-O- [beta] -D
-glucoside (7.813) 9.92 ± 1.68 6.80 ± 0.58

As shown in FIG. 2 and Table 2, the retention time of stilbene glucoside purely isolated from red seaweed was 7.813 minutes, and the correlation values were all higher than 0.999. The content of stilbene glucoside in the combined extract was 6.80%.

Example  3: Identification of effects on the survival and proliferation of neurons in the complex extract

3-1: Hippocampal cells HT22 Identification of protection against

To investigate the protective function of complex extracts on hippocampal cell related to memory and cognitive function improvement, we examined the effect of complex extracts on glutamate toxicity (excitotoxicity) by treating glutamate on hippocampal HT22 cells. Specifically, HT22 cells were pretreated with the combined extract prepared in Example 1 at 0.01, 0.1, 1 and 10 / / ml for 24 hours, and the cytotoxicity induced by glutamate was used for growth and proliferation of cells (MTT) and lactate dehydrogenase (LDH) assay, which is an assay method for cell damage. The results are shown in Fig.

As shown in FIG. 3, the glutamate treatment decreased the cell survival rate by about 40% as compared with the control group. However, the cell survival rate was increased depending on the treatment with the complex extract of the present invention, and the result of the lactate dehydrogenase assay was also inhibited.

3-2: Identification of effects on neural stem cell proliferation

The proliferative effects of C17.2 neural progenitor cells (NPCs), which play an important role in the recovery of central nervous system function, were investigated by MTT assay. Specifically, the complex extract prepared in Example 1 was pre-treated for 24 hours with C17.2 neural stem cells, and cell survival analysis was performed. The results are shown in FIG.

As shown in FIG. 4, cell proliferation was significantly increased at a concentration of 10 ng / ml to 10 μg / ml of the complex extract of the present invention.

Example 4: Tau (Tau) the expansion of the inhibitory action of the compound of the extract protein hyperphosphorylation Chemistry

Hyperphosphorylation of tau protein is known to be the cause of Alzheimer's disease, one of the degenerative neurological diseases. We investigated whether the compound extract has the inhibitory effect on tau hyperphosphorylation through the ex vivo system using cerebral cortex. LiCl was used as an experimental control with GSK3? Inhibitor. The results are shown in Fig.

As shown in FIG. 5, when 1 mg / ml of the complex extract of the present invention was applied to the cerebral cortex, the expression of phosphorylated tau was markedly reduced compared to GSK3? (+). That is, it was observed that the compound extract of the present invention has an inhibitory effect on tau protein phosphorylation.

Example  5: Cerebral blood flow  Identification of the effect of multiple extracts on growth

It is known that decrease of cerebral blood flow leads to decrease of brain metabolism and decrease of cognitive function. It is known that activity or drug which increases cerebral blood flow has a positive effect on memory and cognitive function. PeriFlux Laser Doppler Flowmetry System was used to slowly inject 3, 10, 30, 100, 300 and 1000 μg of the compound extract prepared in Example 1 through a femoral vein of an experimental animal (C57BL / 6J mouse) The cerebral blood flow at the middle cerebral artery region was measured. The results are shown in FIG.

As shown in FIG. 6, the cerebral blood flow was significantly increased by 30% or more depending on the concentration of the complex extract of the present invention.

Example 6: Confirmation of the effect of complex extract on light-induced thrombosis ( photothrombosis )

The vascular central nervous system disorders such as cerebral infarction are very closely related to memory and cognitive ability, and the possibility of development due to more serious dementia is very high. Therefore, we examined the effect of compound extract using a mineral oil thrombosis mouse model. 500 mg / kg of the combined extract prepared in Example 1 was orally administered to experimental animals (C57BL / 6J mice) once a day for 3 days, and the results are shown in FIG. As shown in FIG. 7, the complex extract-treated group of the present invention showed marked decrease in the area of cerebral infarction compared to the control group, and edema was similarly decreased.

Example 7: Effect of Compound Extract on Improvement of Memory and Cognitive Function of Normal Animals

7-1: Hazard test of compound extract

Body weights were measured during oral administration of 100 mg / kg and 500 mg / kg of the complex extract prepared in Example 1 for 6 weeks to 6-week-old experimental animals (C57BL / 6 mice) .

As shown in FIG. 8, no significant difference was observed between the compound extract-treated group of the present invention and the vehicle. In other words, high concentration was used due to its characteristic of extract, but the harmfulness due to the diet was not shown in the complex extract of the present invention.

7-2: Effect of compound extract on memory and learning ability

Morris water maze analysis was performed to examine the effect of dietary supplementation on memory and learning ability. Morris water maze analysis is used as a behavioral test to improve memory and cognitive function. It is a behavioral test that examines spatial memory and learning ability. Finds a hidden platform, while a person with a cognitive impairment wanders without finding a target.

The animals were fed for 2 weeks. The experimental animals were randomly placed in four directions of the round pool, and then trained to find hidden platforms. Through the image tracking system, the results were collected. Followed by a total of 5 days. The results are shown in Figures 9, 10, and 11.

As shown in FIG. 9 and FIG. 10, in the complex extract-treated group of the present invention, there was no abnormality in movement or exercise ability because there was no significant difference in distance moved and swimming velocity during the training period Could know. The five - day training showed that the total distance traveled and the time to reach the hidden platform gradually decreased, recognizing and remembering the hidden platform, unlike the disease - induced animal models or hippocampal damage animals. As shown in FIG. 11, in the case of the latency time for finding the target, the complex extract of the present invention finds the hidden platform significantly faster and the complex extract has the spatial perception ability and the cognitive function .

7-3: Effects of compound extracts on short-term learning and memory

In order to observe short-term learning and memory, two-week combined extracts were administered and passive avoidance test was performed. Passive avoidance test is an experiment that can check hippocampal dependent short term learning & memory. Measures the time taken from the bright room to the dark room, and if the cognitive function is impaired, the potential time is significantly reduced and memory retention can be measured

Specifically, the passive avoidance test was carried out in a box divided into a partition with a grid through which a current can flow in the floor, as a behavioral test using a habit where an experimental animal preferred a dark space. On the training day, when animals were placed in bright compartments, they entered dark compartments, which automatically closed the door and gave an electric shock of 0.25 mA for 2 seconds. Experiments were conducted 1 day and 6 days after the training. The experimental animals were placed in the illuminated compartment, and the animals were crossed over to the dark compartment for 300 seconds to measure the cross-over latency. Animals that last 300 seconds in the bright compartment mean improved learning and memory. The results are shown in Fig.

As shown in Fig. 12, all the groups of experimental animals entered the dark room on the training day, and there was no difference in the cross-over latency time between the groups. On the 1st day after the training, the delayed time was significantly increased in the group administered with the compound extract of the present invention compared with the control group, and it was found that it helps to improve the short term memory.

Example 8: Effect of compound extract on function improvement of memory and cognitive decline model

Aβ _25-35 fragment was injected intracerebroventricular injection (icv) into experimental animals (C57BL / 6J mouse), and Aβ peptide accumulates in the brain 4 weeks later, causing cognitive impairment symptoms. The combined extracts of 100 mg / kg and 500 mg / kg of the extract prepared in Example 1 were orally administered to the animal models with a reduced cognitive function for 4 weeks at a fixed time of about 10 am, The results are shown in FIG.

As shown in FIG. 13, in the group administered with the compound extract of the present invention, significant enhancement effect was observed in the concentration-dependent learning performance.

Claims (9)

Wherein said brain or neurological disease is selected from the group consisting of cerebral infarction, ischemic brain disease, Alzheimer ' s disease, memory < RTI ID = Disorder, cognitive disorder, and learning disorder. [Claim 4] The method according to claim 1, wherein the complex extract is prepared by mixing the extract of Red Seaweed, Sulfur, Sodium Chloride, and Seokjangpo with water, an alcohol having 1 to 5 carbon atoms, acetone, ethyl acetate, butyl acetate, chloroform, hexane, diethyl ether, And a mixed solvent thereof. The pharmaceutical composition for preventing or treating brain diseases or neurological diseases according to claim 1, Wherein the brain or neurological disease is selected from the group consisting of cerebral infarction, ischemic brain disease, Alzheimer's disease, memory disorder , Cognitive disorders, and learning disorders. [Claim 4] The method according to claim 3, wherein the complex extract is obtained by mixing an aqueous solution of sodium chloride, sulfuric acid, ground water, and silica gel in water, an alcohol having 1 to 5 carbon atoms, acetone, ethyl acetate, butyl acetate, chloroform, hexane, diethyl ether, And a mixed solvent thereof. The food composition according to any one of claims 1 to 3, A food composition for enhancing brain or cognitive function, comprising an effective component of a compound extract of red sea bream, red sea bream, rhododendron, rosemary, and safflower. The food composition according to claim 5, wherein the brain or cognitive function is memory, thinking, comprehension, calculation, learning, judgment or concentration.

The pharmaceutical composition for preventing or treating brain diseases or neurological diseases according to claim 1, wherein the compounding ratio of the above-mentioned sodium hypochlorite, sulfuric acid, groundnut, and stearic acid is 1: 20: 0.5-10: 0.1-5: Gt; The food for preventing or ameliorating brain diseases or neurological diseases according to claim 3, wherein the compounding ratio of the above-mentioned sodium hypochlorite, persimmon, persimmon, and safflower is from 1 to 20: 0.5 to 10: 0.1 to 5: Composition [6] The food composition according to claim 5, wherein the mixing ratio of the at least one selected from the group consisting of sodium hypochlorite, hyaluronic acid, groundnut oil and safflower is from 1 to 20: 0.5 to 10: 0.1 to 5: 0.1 to 5.

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